Aqueous two phase partitioning of fish proteins: partitioning studies and ATPS evaluation

Abstract

A suitable Aqueous Two Phase System (ATPS) has been identified for the partitioning of crude fish proteins from fish processing industrial effluent. A detailed study has been performed to analyze the influence of various parameters on protein partitioning such as molecular weight of PEG, effect of different salts (MgSO4_, K₂HPO_4, Na₃C₆H₅O_7, Na₂SO_4, (NH₄) ₂PO_4, K₃C₆H₅O₇) and their concentrations, pH, temperature, Tie Line Length (TLL), effluent loading and volume ratio. PEG 2000 - sodium sulphate ATPS was found to be a most favourable system among the selected ATPS for higher partition coefficient of proteins. The binodal curve and equilibrium characteristics of PEG 2000 - sodium sulphate were established and fitted to empirical equations. The equilibrium compositions (tie line) were correlated using Othmer–Tobias and Bancroft equations.

Introduction

The effluent generated from fish process industry contains a high organic load and it should be treated/recovered before discharging, in order to prevent a negative impact on the environment. On the other hand, this effluent contains a large amount of potentially valuable proteins. The degree of effluent generation from fish processing industry depends on type of operation involved and amount of seafood processed. Typically, a plant of 100 t/h fish processing capacity plant generates 10–40 m³/h effluent with protein loads of 0.5–20 g/l (Mateusz and Daniela 2009; Maria and Rodrigo 2002; Maria et al. 2004). Research has been carried out in order to develop appropriate methods to convert these wastes into useful products, like fish protein hydrolysates. The fish protein hydrolysates have been reported to exhibit various bio functionalities such as antioxidative, antibacterial, anti hypersensitive and anticancer properties etc. (Kim and Mendis 2006). Recovered protein molecules have vital importance in several pharmaceutical industries as well as feed formulations apart from providing value addition to fish processing industrial waste. Conventional methods used for recovery and purification of proteins from fish processing industrial effluent include enzymatic hydrolysis, centrifugation (Guerrerof et al. 1998), filtration, flocculation, precipitation (Taskaya and Jaczynski 2009), pH shifting (Helgi and Ingrid 2009), membrane filtration (Mateusz and Daniela 2009; Maria and Rodrigo 2002) and emerging technologies such as subcritical water hydrolysis, supercritical fluid extraction and ohmic heating (Kobsak et al. 2008). However the proteins purified through these conventional techniques may lead to poor functionality and nutritive value (Sanmartin et al. 2009) and high production cost. Therefore, it is essential to design a reliable separation strategy that would improve yield, selectivity, economy and feasibility of the proteins purification process.

Aqueous two-phase systems (ATPS) have been successfully used for separation and purification of proteins, metal ions, nanoparticles and dyes from various sources such as waste water, fermentation broth, animal and plant cell organelles (Selvaraj et al. 2011). ATPS are formed by adding two water-soluble polymers or polymer and salt together above a critical concentration. The ATPS have advantages over other conventional separation techniques due to high water content in both the phases, offering high biocompatibility and low interfacial tension which results high mass transfer and minimal degradation of biomolecules. ATPS also provides high yield with relatively high handling capacity. Polymer/salt ATPS are more preferred over polymer/polymer systems since it is easy to separate the phases due to high density difference exist between the phases. In addition, the system can be easily scaled up. ATPS could be a good alternative for a first purification step since such systems allow removal of several contaminants by a simple and low-cost process (Albertsson 1986; Raghavarao et al. 1995 and 1998; Rajni 2001; Rosa et al. 2010 and 2011).

The mechanism governing the partition of biomolecules from a particular source in ATPS is still not fully understood and largely unknown. In general, protein partitioning is driven by Van der Waals, hydrophobic, hydrogen bond, and ionic interactions between the biomolecules and the surrounding phase. Hence the partition may be influenced by the concentrations and molecular weight of phase-forming polymer, type and concentration of phase-forming salt, and pH. Several studies have resolute on the influences of the above mentioned parameters on the separation of various biomolecules. Tubio et al. (2007) has studied the effect of different factors such as PEG molecular weight, pH, TLL, temperature and the presence of an inorganic salt on the protein partition coefficient. Chia-Kai and Been (2006) has studied extraction behaviour of lysozyme from chicken egg white by PEG - sulphate system and found that the maximum partitioning was occurred at a temperature of 25.8 °C and pH 10. Shahbaz and Omidinia (2008) reported the influence of PEG molecular weight and its concentrations, pH, ammonium sulphate concentrations and TLL on recombinant enzyme phenylalanine dehydrogenase partitioning. Silva et al. (2007) studied the influence of PEG 1500 with various type of salts (potassium phosphate, sodium citrate, lithium sulphate or sodium sulphate) and temperature (5–45 °C) on the caseinomacropetide partition coefficient and the thermodynamic parameters (ΔH^o, ΔS^o and ΔG^o) were calculated for PEG1500 and sodium citrate ATPS at different PEG concentrations. The crude proteins (Zea mays malt) were partitioned from maize malt using PEG 6000/CaCl₂ system and the influence of phase composition and pH on the partition coefficient were studied and optimized through statistical method (Ferreira et al. 2007). Further the α- and β-amylase enzymes from Zea mays malt were purified 130-fold in the salt rich phase by continuous extraction in a PEG/CaCl₂ ATPS (Biazus et al. 2007).

Nowadays, ATPS have been utilized to recover valuable bio-molecules from various industrial wastewaters. Saravanan et al. (2007) studied the recovery of proteins from tannery wastewater using PEG 4000–magnesium sulphate. The same research group (2006) has also reported the recovery of value-added globular proteins from tannery wastewater using PEG 6000 - sodium sulphate salt ATPS. Perumalsamy and Murugesan (2012) developed an environmentally benign PEG-sodium citrate ATPS for the recovery of value added cheese whey proteins from dairy effluents. Recently, Regupathi et al. (2012) developed and studied a new PEG/lithium citrate salt based ATPS and applied to the fish industry effluent to partition the proteins. The ATPS formed with PEG (4000, 6000 and 8000) with salts (tri-potassium citrate, tri-sodium citrate, sodium sulphate and lithium citrate) were studied and found that the PEG 4000 - lithium citrate ATPS resulted with maximum partition coefficient of 7.82 at TLL 38 %. However the usage of lithium salts at higher concentrations has been toxic (Aral and Vecchio-Sadus 2008). Lithium in water is a strong base and reacts slowly with water and liberates hydrogen, a flammable gas and generates the lithium hydroxide in the solution. Lithium compounds are generally pyrophonic and require special handling. Therefore the salt rich bottom phase need to be treated before its disposal to environment.

Accordingly the present work was formulated to identify an environmentally suitable, best ATPS to partition the crude proteins from the fish processing industry effluent by considering PEG (2000) - salts (MgSO_4, K₂HPO_4, Na₃C₆H₅O_7, Na₂SO_4, (NH₄) ₂PO_4, K₃C₆H₅O₇) ATPS systems and varying their concentrations at different pH. The effect of PEG molecular weight, temperature, effluent loading and volume ratio were studied. The binodal curve and tie lines were developed and studied at 25 °C for the PEG2000- Na₂SO₄.

Materials and methods

MgSO_4, K₂HPO_4, Na₃C₆H₅O_7, Na₂SO_4, (NH₄) ₂PO₄ and K₃C₆H₅O₇ salts of purity analytical standard (PAS) were obtained from Merck, Mumbai, India. Analytical grade Polyethylene Glycol (PEG) of average molecular weight 1.9 kDa, 3.85 kDa, 5.8 kDa and 7.75 kDa (PEG 2000, PEG 4000, PEG 6000 and PEG 8000) was purchased from Sigma Chem. Co., USA. All the reagents were of analytical grade. Double distilled water was used for the present work. Bradford reagent, (Sigma Aldrich Inc., Germany) was used to estimate the protein content of the equilibrium phases and effluent. The fish effluent used in the experiments was obtained from a fishmeal processing industry located at Mangalore, India.

Preparation of ATPS

The PEG molecular weight their respective concentration and salt concentration were chosen based on the phase diagrams. To study the effect on partitioning of protein from fish processing effluent, different salts like MgSO_4, K₂HPO_4, Na₃C₆H₅O_7, Na₂SO_4, (NH₄) ₂PO_4, and K₃C₆H₅O₇ and their concentrations were mixed with varying PEG (2000, 4000, 6000 and 8000), fish processing industry effluent 50 % (w/w) and distilled water were used to attain 100 % (w/w) of the system. The mixtures were dissolved completely using vortex mixer. Phase separation was achieved by centrifugation for 10 min at 3,500 rpm. The samples were incubated to attain equilibrium in a thermostat maintained at a constant temperature of 25 °C for 24 h with an uncertainty of ± 0.05 °C (Lab. Companion, Model RW-0525G, Korea). The top phase was carefully separated using a pipette. Volumes of the separated phases were measured and aliquots from each phase were taken for further analysis.

Measurement of polymer and salt concentrations

Determination of PEG concentration was performed by measuring refractive index using Digital refractometer (RX-500, ATAGO Co. ltd, Japan). Calibration curves were prepared with known concentrations of PEG and Salt in the homogeneous regions of binodal diagram of the individual ATPS. The relation between the refractive index (n_D) and the mass fraction of polymer (w_p) and salt (w_s) is given by Eq 1.

$${\mathrm{n}}_{\mathrm{D}}={\mathit{a}}_0+{\mathit{a}}_1{\mathit{w}}_{\mathit{P}}+{\mathit{a}}_2{\mathit{w}}_{\mathit{S}}$$ 1

Where a₀, a₁ and a₂ are the fitting parameters.

Salt concentrations in the top and bottom phase were measured using flame photometer. The phases were diluted appropriately to the required concentrations that are suitable for the analysis in flame photometer. Salt calibration curve was prepared with known concentration of salt with suitable dilutions.

Protein estimation

Bradford method was used to estimate total protein concentration. The samples were read at 595 nm against the blanks with the same compositions of PEG and salt. Assays were performed in triplicate and their respective averages were used in the calculations. The results are expressed in terms of bovine serum albumin (BSA) equivalents.

Partition coefficient (K) was calculated by (Eq. 2);

$$\mathrm{K}=\frac{{\mathrm{C}}_{\mathrm{tp}}}{{\mathrm{C}}_{\mathrm{bp}}}$$ 2

Partition coefficient is defined as the ratio of protein concentration in top phase, C_tp (mg/ml) to bottom phase, C_bp (mg/ml). Phase volume ratio (VR) is defined as the ratio of volume of top phase, V_tp (ml) to that of bottom phase, V_bp (ml) (Eq. 3).

$$\mathrm{VR}=\frac{{\mathrm{V}}_{\mathrm{tp}}}{{\mathrm{V}}_{\mathrm{bp}}}$$ 3

The percentage yield of protein in the top phase is calculated using Eq 4.

$$\mathrm{Yield}\left(\%\right)=\frac{{\mathrm{C}}_{\mathrm{tp}}\times {\mathrm{V}}_{\mathrm{tp}}}{{\mathrm{C}}_0\times \mathrm{V}{}_0}\times 100$$ 4

Where, C_o is initial concentration of protein (mg/ml), V_o = volume of feed sample (ml).

Phase diagram

Binodal curve for PEG 2000 and sodium sulphate ATPS was developed through could point method. The experiment was conducted in 250 ml jacketed vessel at 25 °C with an uncertainty of ± 0.05 °C. A known concentration of salt solution was titrated against to polymer solution or vice versa, until the clear solution turn out to be turbid. The phase composition at this point was estimated through weighing method using a higher precision (±0.01 mg) analytical balance. Further known quantity of water was added until the turbidity get into clear solution. This procedure was repeated to obtain different bimodal points (Regupathi et al. 2011 and 2012).

The equilibrium characteristic of the system was analysed through tie lines. Pre weighted quantity of varied PEG 2000, sodium sulphate and water were added into a centrifuge tube of 15 ml to obtain 10 g system. The samples were thoroughly mixed and centrifuged at 3500 rpm for 10 min. The mixture was allowed to settle for 24 h at 25 °C in a temperature bath, resulted in to two equilibrated phases. The individual phase volumes were measured and subsequently separated using micro pipette. The concentrations of PEG 2000 and sodium sulphate in both top and bottom phase were determined using refractive index and flame photometer, respectively, which represents the tie line composition. Further the tie line length was calculated using the equilibrium compositions by Eq. (5).

$$\mathit{TLL}={\left[{\left({\mathit{W}}_{\mathit{P}\left(\mathit{T}\right)}-{\mathit{W}}_{\mathit{P}\left(\mathit{B}\right)}\right)}^2+{\left({\mathit{W}}_{\mathit{S}\left(\mathit{T}\right)}-{\mathit{W}}_{\mathit{S}\left(\mathit{B}\right)}\right)}^2\right]}^{\begin{array}{l}1/2\\ \end{array}}$$ 5

Results and discussion

Selecting a suitable ATPS for the higher partitioning of proteins in a specific effluent is a complex process since it is highly depends on the characteristics of the solute such as hydrophobicity, molecular size, electrochemical property, molecular conformation and bio specificity as well as the ATPS’s properties such as phase forming polymer or salt, pH, temperature, TLL, effluent loading and volume ratio. Initially the suitable salt (at fixed concentration) was identified by conducting the experiments at different PEG2000 concentration and varying salt concentration at fixed PEG2000 concentration. Further the effect of pH, PEG molar mass, temperature, and volume ratio were studied for the selected system.

Effect of PEG 2000 concentrations with different salts

The partitioning of soluble crude proteins from fish processing industrial effluent was studied by varying the PEG2000 concentration at a fixed salt concentration of 22.5 % (w/w). Five different salts as MgSO_4, K₂HPO_4, Na₃C₆H₅O_7, Na₂SO_4, (NH₄) ₂PO₄ and K₃C₆H₅O₇ were considered for the present work (Fig. 1). From the figure it was observed that the sodium sulphate and potassium di-hydrogen phosphate ATPSs showed maximum partitioning coefficient in the range of 1.7 to 2.3. The partitioning coefficient found to increases with increasing PEG2000 concentration for all the selected salts due to salting out effect except ammonium phosphate and resulted in movement of proteins from bottom phase to top phase. At higher PEG concentration, the degree of protein interaction with PEG found to increases due to the hydrophilic nature of the system. Saravanan et al. (2006 and 2007) has observed similar effect for tannery waste contained proteins. However, there is a limitation in the solubility of protein in the top phase of PEG2000 - (NH₄) ₂PO₄ system at higher PEG concentration; hence the partitioning coefficient was found to decrease with increasing PEG concentration. However MgSO₄ ATPS showed maximum precipitation at the inter phase. The mixed effect was observed probably due to the combination of hydrophobic, ionic change and free volume interactions exist with the proteins. The affordable partition coefficient was observed at a PEG2000 concentration of 15 % (w/w) in all the selected salts forming ATPS. Mokhtarani et al. (2008) also conducted similar experiments for the partitioning of Ciprofloxacin and reported that the salt concentration had significant effect than the PEG concentration.

Fig. 1.

Effect of PEG2000 concentrations with different salts (∆ Na₂SO₄, □ K₂HPO₄,◊ (NH₄) ₂PO₄, × K₃C₆H₅O₇ and ○ Na₃C₆H₅O₇) of fixed concentration 22.5 % (w/w) on the partition coefficient at 25 °C

Selection of phase forming salt

The experiments were carried out at constant PEG2000 concentration of 15 % (w/w) with different salts and their concentrations to select the suitable ATPS for efficient fish protein partitioning (Fig. 2). The ATPSs are formed for the said purpose with 50 % (w/w) of fish processing industrial effluent. From the figure it was observed that the partitioning coefficient was increasing with increasing salt concentration due to the decrease in the solubility of protein in bottom phase. The free water volume available in the bottom phase for the protein dissolution may decreases as the salt concentration increase. However the top PEG phase shows stronger affinity towards the proteins, due to the corresponding increase in the PEG concentration in the top phase. Further, the partition coefficient may vary for different salts as shown in Fig. 2 due to the nature of the salt cation and anion and the system’s net charges. The influence of salt on the partitioning is caused by the nonuniform distribution of the salt ions in the upper and lower phases and by the difference in the electric potential, which improves the movement of the protein to the other phase through electrostatic repulsion/attraction. The said effect may be explained through the Hofmeister series (Gupta et al. 2002), which is reported as, for anions SO₄²⁻ > HPO₄²⁻ > CH₃COO⁻ > Cl⁻ > Br⁻ > I⁻ > SCN⁻, and for cations Li⁺ > Na⁺ > K⁺ > NH₄⁺ > Mg²⁺. At higher concentrations of the salt, the ions to the left of the series decrease the protein solubility (salting-out effect) in the salt rich bottom phase. Further the hydrophobic interaction between the protein and polymer phase increases due to the hydration effect of the salt molecule surrounding the protein and lead to the aggregation of proteins in the top phase. The present result shows the similar effect for the selected salt systems. Among the salt systems potassium phosphate and sodium sulphate showed better partition when compared to ammonium phosphate, sodium citrate and potassium citrate salts. A similar behaviour had been reported earlier for various proteins in different systems (Saravanan et al. 2006; Kohler et al. 1991; Perumalsamy and Batcha 2011).

Fig. 2.

Effect of salts (Δ Na₂SO₄, □ K₂HPO₄, ◊ (NH₄) ₂PO₄, × K₃C₆H₅O₇ and ○ Na₃C₆H₅O₇) concentration on partition coefficient at 15 % (w/w) PEG 2000 concentration at 25 °C

Effect of pH on protein partitioning

In general, the pH affects the partitioning of protein by changing the net charge of the phases, consequently the hydrophobic and hydrophilic forces exist in the system may be altered and which promotes the partitioning of the proteins towards a particular phase. However the change in electrostatic force with pH may vary significantly for different salts. Hence in the present study the pH was altered in the range of 5 to 10 for all the ATPSs (with different salts) to understand the partitioning behaviour of the systems with constant PEG2000 and salt concentration (Fig. 3). From the figure it was observed that a small change in pH values had a stronger effect on partition coefficient in all the ATPS. Significant increase in partition coefficient as well as top phase yield was observed for all the systems around the pH values of 5 to 6. However the maximum partition coefficient of 13.14 was observed at pH 5 for PEG 2000 - sodium sulphate ATPS. At lower pH, the hydrophobic attraction between PEG and proteins were predominant due to the net negative charge gained by the protein molecules and resulted to higher partitioning coefficient. Conversely the partitioning coefficient was found to decrease with increasing pH since the bottom phase was enriched with sulphate which reduces the net hydrophobic interaction. The ATPS with sulphate as the phase-forming salt shown higher partition since the sulphate anion has the ability to promote hydrophobic difference between the phases through salting-out phenomena (Chia-Kai and Been 2006). Similar behaviour was observed in the literature for other systems (Patil and Raghavara 2007; Ferreira et al. 2007; Yucekan and Onal 2011; Perumalsamy and Batcha 2011). The partitioning coefficient of proteins from Shrimp waste increases when the pH increases from 6 to 8 and maximum partition coefficient of 2.96 was achieved at pH 8 (Ramyadevi et al. 2012). The results revealed that the fish proteins gained maximum negative charge at the pH of 5. From the observations, it was decided to carry out the further experiments at 15 % (w/w) of PEG 2000 and 22.5 % (w/w) sodium sulphate and at an optimised pH of 5 for the purification of proteins from fish processing industrial effluent.

Fig. 3.

Effect of pH on partitioning coefficient for different salts (Δ Na₂SO₄, □ K₂HPO₄, ◊ (NH₄) ₂PO₄, × K₃C₆H₅O₇ and ○ Na₃C₆H₅O₇) with 15 % (w/w) PEG concentration at 25 °C

Effect of PEG molecular weight on protein partitioning

Molecular weight of PEG alters the partition coefficient of biomolecules, since the hydrophobic interaction and effective excluded volume of the ATPS greatly depends on it. Figure 4 shows the effect of molecular weight on the partitioning of soluble proteins from fish processing industrial effluent in 15 % (w/w) of PEG and 22.5 % (w/w) sodium sulphate ATPS at 5 pH. The partition coefficient of proteins decreased with increasing PEG molecular weight. The increase in PEG molecular weight increases the hydrophobicity and effective excluded volume of polymer rich top phase in the systems which favours the partitioning of proteins in the PEG phase however the reduction in top PEG rich phase free volume for biomolecule salvation reduces the protein partitioning in top phase. Similar observations were reported for the partitioning of BSA in PEG-tri sodium citrate/ tri potassium citrate system (Perumalsamy and Batcha 2011; Kalaivani and Regupathi 2013) [13, 15]. However, the increase of PEG molecular weight associated with increase in the hydrophobicity of PEG, does not favour the partitioning of the fish protein in to the top PEG rich phase which indicates that the Effective Excluded Volume of PEG predominates the hydrophobic effect. Further, at higher PEG molecular weight the proteins start to precipitate at the interface due to the significant reduction in free volume for biomolecule solubility in both the phases. The maximum partition coefficient 13.14 and 84.45 % yield were obtained at 15 % (w/w) PEG 2000–22.5 % (w/w) sodium sulphate ATPS at pH 5. Saravanan et al. (2007) has also reported a similar result for the recovery of proteins from tannery wastewater in PEG-magnesium sulphate system. Ramyadevi et al. (2012) observed that the partition coefficient and extraction yield of proteins from Shrimp waste was decreased with the increase of PEG molecular weight from 4000 to 10,000.

Fig. 4.

Effect of PEG molecular weight on protein partitioning on PEG 15 % (w/w) - sodium sulphate 22.5 % (w/w) at 25 °C. (□) K and (○) yield %

Effect of temperature on protein partitioning

To select the optimal temperature for the process study, different temperatures of 20–50 °C at constant pH 5 were studied and the results are shown in Fig. 5. The change in the partition coefficients of the biomolecules with temperature may be attributed towards variation in the phase compositions. In the present study, the obtained results indicated that increase in temperature leads to decrease in partition coefficient of protein. The maximum protein partition 14.13 was observed at 25 °C with yield of 84.45 %. At higher temperature, PEG structure becomes more extended and as a result the preferential interaction between proteins and PEG decreases, consequently the partition coefficient and yield of proteins extracted also decreases. On the other hand, increasing the temperature the proteins denature and lose its activity. Similar temperature effect were found for various biomolecules such as globular proteins from tannery wastewater (Saravanan et al. 2006 tannase from fermentation broth (Naidu et al. 2008), alkaline phosphatase from Bacillus licheniformis (Pandey and Banik 2010), bromelain from pineapple (Ketnawa et al. 2010), BSA (Perumalsamy and Batcha 2011) and β-glucosidase from Trichoderma reesei (Gautam and Simon 2006).

Fig. 5.

Effect of temperature on protein partitioning in 15 % (w/w) PEG 2000–22.5 % (w/w) sodium sulphate. (□) K and (○) yield %

Effect TLL on protein partitioning

In general PEG and salt concentrations present in the ATPS were mainly responsible for the two phase formation. However, the type and nature of the salt present in the system also contributed towards protein partitioning. The TLL %, which in turn represents the equilibrium of the ATPS, was utilized to study the combined effect of the weight fraction of PEG and salt. The effect of TLL on partition and yield in PEG 2000 - sodium sulphate at 25 °C are shown in Fig. 6. It was observed that as the TLL increases the partition coefficient also increases. At larger TLL %, PEG concentration in the top phase and salt concentration in the bottom phase increases. Due to increase in PEG concentration in top phase, the number of polymer-protein hydrophobic interactions may increase. On the other hand, the biomolecules may be excluded from the bottom phase to top phase due to the solubility limits of the biomolecule in salt phase through effective salting out phenomena. It promotes the partition of protein from the bottom phase to top phase. The similar phenomenon has been reported by Patil and Raghavara (2007) and Ferreira et al. (2007). Ramyadevi et al. (2012) also reported that the partition coefficient of proteins from Shrimp waste was increased with increasing TLL and found maximum of 2.96 at a TLL of 36.

Fig. 6.

Effect of TLL on protein partitioning coefficient and yield in PEG 2000 - sodium sulphate system

Effect of fish effluent loading on protein partitioning

As described in the earlier sections, fish processing effluent load of 50 % (w/w) was used. Higher effluent loads would be advantageous since larger feed volumes could be processed in single stage ATPS. Fish processing industrial effluent loading is considered to be one among the important variable in the selection of partitioning system and was used to examine the processing capacity of ATPS by varying effluent loading. Effluent loading of 10–60 % (w/w) was used and their respective loading effects are shown in Fig. 7. As the effluent loading increased from 20–50 % (w/w) gradually, partition coefficient increases from 2.8 to 10.62 and yield from 61.05–85.28 % respectively. Further loading at 60 % (w/w) the partition coefficient decreased to 3.36. This is due to increased protein precipitation at interphase and due to insufficient free volume available in the phases. Similar observations of precipitation and phase’s free volume at higher loading of proteins to the system were reported in the case of biomass containing fermentation broths (Patil and Raghavara 2007; Selvakumar et al. 2012).

Fig. 7.

Effect of fish effluent loading on protein partitioning coefficient and yield in PEG 2000 - sodium sulphate system

Effect of volume ratio on protein partitioning

The important step of any purification process should combine in lower volume and high yield with a partition of the target molecule. In ATPS this could be easily achieved by altering volume of the phases, where the target molecule is partitioned into one particular phase. Volume ratio was varied by changing the volume of the individual phases at constant total volume in which the phase compositions (PEG and salt) of the top and bottom phases remain same with respect to their tie tiles. The differential partitioning coefficient of fish proteins at different phase volume ratios are shown in Fig. 8. The maximum salvation of the proteins in the individual phases will be dictated by the solubility limit of the target compound in the accommodating phases. At lower volume ratios the protein solubility limits was reached in the bottom phase. However the remaining proteins try to move towards the top phase and dissolved in the excess free volume available, which in turn leads to the increase in the partitioning coefficient. Moreover, it was suggested that as the volume ratio increases, the protein partition coefficient increases wherein the desired molecule gets transferred to the polymer rich top phase as described in other literatures; betalains partition coefficient in PEG 6000 - ammonium sulphate (Chethana et al. 2007), chymosin and pepsin partition coefficient in PEG - phosphate (Spelzini et al. 2006) and penicillin acylase partition coefficient in PEG - sodium citrate (Marcos et al. 1998) ATPS. The increase in partitioning coefficient was noticed till the saturation limits attained by the PEG top phase, there after the proteins were precipitated at the inter-phase. Hence the yield was observed to increase till the volume ratio of 2.5 and further remains constant with volume ratio (Fig. 8).

Fig. 8.

Effect of volume ratio on protein partitioning coefficient and yield in PEG 2000 - sodium sulphate system

Phase diagram for PEG 2000 - sodium sulphate

The overall partitioning behaviour was primarily depends on the equilibrium characteristics of the ATPS. The phase composition and their equilibrium were expressed through the phase diagram of the ATPS, which includes the binodal curve and tie lines. The phase diagram describes the boundary between two-phase and single phase region and provide the equilibrium concentrations of the phase components at the given feed composition. Hence the phase diagram was developed at the maximum protein partitioning conditions (25 °C and pH 5) for PEG 2000 - sodium sulphate ATPS.

Binodal curve

The binodal curve for the selected PEG 2000 - sodium sulphate ATPS was obtained through could point method (Fig. 9). Further, the experimental binodal data were fitted by modifying the constants and coefficients of the expressions available in the literature equations (Cheluget et al. 1994; Zafarani-Moattar and Hamidi 2003; Regupathi et al. 2011). The constants and coefficients with standard deviation were reported in Table 1. Among the equations proposed in literature, the best fit was obtained with the nonlinear Eq 6.

$${\mathrm{W}}_{\mathrm{p}}=\mathrm{a}+{\mathrm{bW}}_{\mathrm{s}}^{0.5}+{\mathrm{cW}}_{\mathrm{s}}+{\mathrm{dW}}_{\mathrm{s}}^2$$ 6

Fig. 9.

Binodal curve and tie lines for the PEG 2000 - sodium sulphate system at 25 °C

Table 1.

Literature empirical correlations and their constants for PEG 2000 - sodium sulphate system at 25 °C

Correlations	a	b	c	D	R2
W p = a + bW 0.5s + cW s (Cheluget et al. 1994 & Regupathi et al. 2012)	0.8472	−3.903	4.443		0.9964
W p = a + bW 0.5s + cW s + dW 2s (Regupathi et al. 2011& Hu et al. 2004)	0.7756	−3.189	2.451	3.570	0.9979
InW p = a + bW 0.5s + cW 3s (Regupathi et al. 2011& Maria et al. 2001)	1.2027	−12.93	26.52		0.8987
$\frac{1}{{\mathit{W}}_{\mathit{s}}}=\mathit{a}+\mathit{b}{\mathit{W}}_{\mathit{p}}^{0.5}+\mathit{c}{\mathit{W}}_{\mathit{p}}$ (Regupathi et al. 2011& Graber et al. 2001)	27.74026	−155.67200	327.53700		0.88610

Where, Wp and Ws are the mass fractions of PEG 2000 - sodium sulphate, respectively. On the basis of the obtained standard deviation (0.997), it was concluded that above mentioned nonlinear equation can be satisfactory used to predict the binodal compositions for the investigated system.

Phase Equilibrium

The phase equilibrium studies of the selected ATPS are necessary for effective development of real system. Equilibrium compositions of the phases are responsible for the partitioning and maintaining the surface properties of targeted biomolecule in ATPS. The equilibrium phase compositions (tie line compositions) and tie line lengths for different feed conditions were obtained at 25 °C and reported in Table 2. The phase diagram, which includes the binodal curve and tie lines for the PEG 2000 - sodium sulphate ATPS, is shown in Fig. 9. The equilibrium phase compositions for different tie lines were correlated using literature empirical correlations of Other – Tobias (eq. 7) and Bancroft (eq. 8).

$$\left(\frac{1-{\mathrm{W}}_{\mathrm{p}}^{\mathrm{t}}}{{\mathrm{W}}_{\mathrm{p}}^{\mathrm{t}}}\right)=\mathrm{K}{\left(\frac{1-{\mathrm{W}}_{\mathrm{s}}^{\mathrm{b}}}{{\mathrm{W}}_{\mathrm{s}}^{\mathrm{b}}}\right)}^{\mathrm{n}}$$ 7

$$\left(\frac{{\mathrm{W}}_{\mathrm{w}}^{\mathrm{b}}}{{\mathrm{W}}_{\mathrm{s}}^{\mathrm{b}}}\right)={\mathrm{K}}_1{\left(\frac{{\mathrm{W}}_{\mathrm{w}}^{\mathrm{t}}}{{\mathrm{W}}_{\mathrm{p}}^{\mathrm{t}}}\right)}^{\mathit{r}}$$ 8

where K, n, K₁, and r are the fitting parameters. Superscripts t and b represent the polymer-rich phase (top phase) and the salt-rich phase (bottom phase), respectively. Subscripts p, s and w stand for PEG, salt and water, respectively. The fitting parameters and the corresponding regression coefficient were reported in Table 3, which can be used to predict the equilibrium composition of the PEG 2000 - sodium sulphate ATPS.

Table 2.

Equilibrium Phase composition of PEG 2000 - sodium sulphate system 25 °C

Feed composition		Top PEG rich Phase		Bottom salt rich phase		TLL % (w/w)
Wp % (w/w)	Ws % (w/w)	Wp % (w/w)	Ws % (w/w)	Wp % (w/w)	Ws % (w/w)
15	9	32.5	0.2	2.7	15.4	34.65
15	10	35.3	1.3	2.9	15.6	36.23
15	12	37.0	0.6	3.4	17.7	39.11
15	15	43.6	1.4	3.7	21.0	45.63
15	18	49.2	3.1	3.9	24.1	50.29

Table 3.

Othmer-Thobias and Bancroft equation constants for the PEG 2000 - sodium sulphate system at 25 °C

Othmer-Thobias equation			Bancroft equation
K	n	R2	K1	r	R2
0.24595	1.25467	0.99110	3.02629	0.81171	0.96788

Conclusion

Aqueous Two Phase Extraction process was experimented to recover soluble proteins from fish processing industrial effluent. The experimental results revealed that the PEG 2000 - sodium sulphate ATPS is a suitable system and maximum partitioning coefficient and yield of fish proteins (14.53 and 97.75 %, respectively) can be obtained at the following conditions: 15 % (w/w) PEG 2000–22.5 % (w/w) sodium sulphate, pH 5, temperature 25 °C, effluent loading 50 % (w/w) and volume ration 3. Further the equilibrium characteristic of the selected ATPS was studied and the respective phase diagram also developed. The obtained results demonstrate the partitioning potential of the selected ATPS, which can be considered as a first step for the crude fish protein purification from the bulk industrial effluent.

Abbreviations

ATPS

Aqueous two-phase systems

TLL

Tie line length (w/w %)

V_bp

Volume of bottom phase (ml)

V_tp

Volume of top phase (ml)

Wp

Weight fraction of polymer

Ws

weight fraction of salt

VR

volume ratio

K

capartition coefficient